This proposal is designed to test the hypothesis that regulation of expression of adenosine deaminase (ADA) is necessary for normal immune functioning. ADA deficiency is an inherited disease responsible for approximately 20% of cases of severe-combined immunodeficiency (SCID). Infants with this disorder are profoundly immunodeficient and usually die from infection or cancer unless they are successfully treated with bone-marrow transplantation. Because transplantation is often unsuccessful in these children, a new alternative, gene therapy into bone-marrow cells, is being considered. Before beginning gene-insertion studies, however, one would like to have data suggesting that the immune defect will be corrected by gene therapy. This issue arises because most gene-transfer vectors have included constitutive promoters for the ADA gene. These vectors will not result in normal ADA regulation. Unfortunately, no one knows whether ADA regulation affects immune development. The proposed research will directly test the hypothesis that regulation of the ADA gene is necessary for normal immune function. Homologous recombination in murine embryo stem cells will be used to create an ADA-deficient animal model. The ADA-deficient mouse will most likely be profoundly immunodeficient as is its human counterpart. The ADA cDNA will be inserted into ADA deficient and normal animals by transgene technology and retroviral gene insertion therapy. The regulation of ADA expression will be altered by inserting the ADA cDNA under the control of different constitutive promoters. The immune function of the animals will be studied with a series of tests designed to cover a broad spectrum of immune responses. Insertion of ADA transgene into a normal animal may affect immune development. Insertion of the transgene into an ADA deficient animal mimics the situation which will occur in human gene therapy. If the immune systems found to be abnormal, this will imply a central role for ADA regulation in the development of normal immune responses. This finding would directly impact on the vector design for ADA gene therapy in humans. On the other hand, if constitutive expression of ADA leads to normal immune functioning, this will encourage investigators to proceed with human studies.